SUMMARY PROJECT 2: Merkel cell carcinoma (MCC) is a rare but often deadly skin cancer caused by the Merkel cell polyomavirus (MCPyV) in 80% of cases. MCPyV T-antigen oncoproteins are persistently expressed in virus- positive MCCs (VP-MCC), while remarkably high numbers of UV-induced neoantigens are detected in virus-negative MCCs (VN-MCC), suggesting both MCC subsets harbor immunogenic epitopes. Based on our early studies of the immune response in MCC, our group led multiple clinical trials that have recently changed the standard of care for this cancer. PD-1 blockade has yielded high response rates in MCC as compared with other solid tumors, and markedly improved outcomes compared to cytotoxic chemotherapy, the only prior option for advanced MCC. Unfortunately, 40% of MCC patients do not initially benefit from PD-1 blockade and ~20% of responders later develop acquired resistance. Consequently, there is an urgent need to identify therapeutically targetable mechanisms of resistance. In this proposal, we seek to identify reversible mechanisms of resistance to PD-1 blockade by analyzing three critical components across this Project: Aim 1: MCC-specific T lymphocytes. We will determine whether T cell infiltration patterns into tumors, T cell clonal diversity, or dysfunctional status are associated with failure to respond to PD-1 blockade therapy. Aim 2: MCC tumor cells. We will determine whether MCC cell intrinsic immune characteristics including expression of additional checkpoint molecules or impaired antigenicity are associated with response/resistance to PD-1 blockade. Aim 3: MCC innate immunity. We will study the correlation between intratumoral infiltration of two innate immune cell types (macrophages and NK cells) and MCC outcome to determine whether therapeutic intervention with innate immune stimulation can augment MCC adaptive immunity. Notably, we have made several significant advances since our May 2017 submission which will greatly increase our ability to identify and test the functional relevance of immune mechanisms mediating resistance to PD-1 blockade. These advances include: (1) A scRNAseq workflow which can be performed on small amounts (1 core biopsy) of cryopreserved material, enabling careful collection and selection of highly informative patient samples and improved depth of analysis; (2) MCC tumor expansion in patient derived xenograft (PDX) mice for subsequent use in functional assays including a slice-culture/explant system. Unbiased approaches including scRNAseq, will be initially employed to optimize our capacity to identify novel and significant immune evasion mechanisms, as we have demonstrated since the time of our initial submission. Under the guidance of our External and Internal Advisory Boards, we will then select the pathways most strongly associated with PD-1 blockade response. These immune evasion pathways will be studied in a larger number of patients and their functional significance/reversibility will be determined. This project will be greatly enabled by our established collaborations with experts in dissecting immuno-oncology mechanisms including Drs. Martin ?Mac? Cheever (FHCRC), Christian Hinrichs (NCI), Drew Pardoll (Hopkins), John Thompson (UW), Suzanne Topalian (Hopkins), John Wherry (UPenn) and Catherine Wu (Harvard).